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Aims of the study The ability of Yersinia enterocolitica strains to form biofilms and the capacity of different alkaloids to inhibit biofilm formation were investigated. Methods and Results The capacity to form biofilm on polystyrene of 31 Y. enterocolitica strains was evaluated. Biofilm and quorum sensing (QS) inhibition of 17 alkaloids were assayed; furthermore, minimum biofilm inhibitory concentration (MBIC) was determined. The capacity to form biofilms among the examined strains seemed to be a strain‐related feature. The best biofilm inhibitors at 100 µmol l−1 were oliverine (1), guatterine (3), liriodenine (4), oliveridine (5) and pachypodanthine (6), which showed biofilm inhibition higher than 87%. Pachypodanthine (6) was the most effective compound with MBIC value of 12·5 µmol l−1 at subinhibitory concentration and also was able to inhibit QS system and reduce yenR expression at this concentration. Conclusion This is the first study to demonstrate that oliverine, liriodenine, and pachypodanthine are able to inhibit biofilm formation of Y. enterocolitica without critically disturbing its growing capacity. At MBIC, pachypodanthine inhibited biofilm formation and QS. Significance and Impact of the Study The use of aporphinoid alkaloids as biofilms inhibitory agents might potentially be useful to treat biofilm‐associated infections in the future.